Transcription factor induced conversion of human fibroblasts towards the hair cell lineage

RESEARCH ARTICLE Transcription factor induced conversion of human fibroblasts towards the hair cell lineage Marı́a Beatriz Duran Alonso1*, Iris Lopez Hernandez1, Miguel Angel de la Fuente1, Javier Garcia-Sancho1, Fernando Giraldez2, Thomas Schimmang1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Duran Alonso MB, Lopez Hernandez I, de la Fuente MA, Garcia-Sancho J, Giraldez F, Schimmang T (2018) Transcription factor induced conversion of human fibroblasts towards the hair cell lineage. PLoS ONE 13(7): e0200210. https:// doi.org/10.1371/journal.pone.0200210 Editor: Olivia Bermingham-McDonogh, University of Washington, UNITED STATES Received: January 31, 2018 Accepted: June 21, 2018 Published: July 6, 2018 Copyright: © 2018 Duran Alonso et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The project was funded by Junta de Castilla y Leon (Project VA024U16, Feder 20142020), Fundacion La Marato (Project 201227-3031), Red de Terapia Celular and Red de medicina regenerativa y terapia celular de Castilla y Leon. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 Instituto de Biologı́a y Genética Molecular, Universidad de Valladolid y Consejo Superior de Investigaciones Cientı́ficas, C/Sanz y Forés 3, Valladolid, Spain, 2 CEXS, Universitat Pompeu Fabra, Parc de Recerca Biomédica de Barcelona, Barcelona, Spain * (MBDA); (TS) Abstract Hearing loss is the most common sensorineural disorder, affecting over 5% of the population worldwide. Its most frequent cause is the loss of hair cells (HCs), the mechanosensory receptors of the cochlea. HCs transduce incoming sounds into electrical signals that activate auditory neurons, which in turn send this information to the brain. Although some spontaneous HC regeneration has been observed in neonatal mammals, the very small pool of putative progenitor cells that have been identified in the adult mammalian cochlea is not able to replace the damaged HCs, making any hearing impairment permanent. To date, guided differentiation of human cells to HC-like cells has only been achieved using either embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, use of such cell types suffers from a number of important disadvantages, such as the risk of tumourigenicity if transplanted into the host´s tissue. We have obtained cells expressing hair cell markers from cultures of human fibroblasts by overexpression of GFI1, Pou4f3 and ATOH1 (GPA), three genes that are known to play a critical role in the development of HCs. Immunocytochemical, qPCR and RNAseq analyses demonstrate the expression of genes typically expressed by HCs in the transdifferentiated cells. Our protocol represents a much faster approach than the methods applied to ESCs and iPSCs and validates the combination of GPA as a set of genes whose activation leads to the direct conversion of human somatic cells towards the hair cell lineage. Our observations are expected to contribute to the development of future therapies aimed at the regeneration of the auditory organ and the restoration of hearing. Introduction Hearing loss is the most prevalent sensorineural deficit in humans, most frequently caused by damage of hair cells (HCs). These are mechanoreceptor cells in the cochlear part of the inner ear and responsible for transducing the information arriving in the form of incoming sound PLOS ONE | https://doi.org/10.1371/journal.pone.0200210 July 6, 2018 1 / 16 Conversion of human fibroblasts to cells expressing hair cell markers Competing interests: The authors have declared that no competing interests exist. waves to the auditory neurons that connect to the brain. Although a small number of inner ear progenitor cells have been identified in neonatal animals that allow for a certain degree of repair following damage, these appear to be dormant in older individuals, which could explain the observed lack of HC regeneration [1,2]. Approaches that could thus be envisaged towards the restoration of hearing are either the in vivo transdifferentiation into HCs of other cell types present in the inner ear (e.g. supporting cells) or transplantation of HC-like cells that have been derived from distinct tissue sources [2,3,4]. HC-like cells have been obtained from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) [5,6,7,8,9,10,11,12,13]. Although the studies where these cells have been more thoroughly characterized have identified properties of vestibular rather than cochlear HCs [9,14], they set the basis for obtaining their auditory counterparts. A promising alternative to the ESCs or iPSCs may be the use of patient-derived somatic cells that, although fully differentiated, can be changed into the desired cell fate while bypassing the full reprogramming process typically undergone by iPSCs [15,16,17,18,19]. This strategy has already resulted in the successful transdifferentiation of fibroblasts, hepatocytes, astrocytes and various differentiated blood cell types into other cell lineages (e.g. cardiomyocytes, neurons, macrophages) [20,21,22,23,24,25,26]. For reprogramming, two main routes have been followed, either the induction of epigenetic changes, or the direct conversion of somatic cells into sought-for lineages through the forced expression of lineage-determining factors [16,18,20,26,27,28,29,30]. Epigenetic alterations have been shown to occur following exposure of the cells to chromatin modifiers such as demethylating agents and histone deacetylase inhibitors, or overexpression of transcription factors such as Sox-2 [31] and Oct-4 [24,32]. This results in the transient expression of sets of genes that are associated with a variety of cell lineages. Subsequent culture of these cells under conditions known to drive the emergence of the cell type of interest in vivo will then promote their differentiation. On the other hand, overexpression of transcription factors associated with a given lineage is thought to result in the direct conversion of the donor cells, through the recruitment of downstream genes and the activation of the corresponding signalling cascades [22,28,29]. Importantly, this approach has already been successfully applied in vivo [2,23,26,33,34]. In the present study, we have adopted a direct conversion approach in order to obtain cells expressing HC markers from cultures of human fibroblasts (hFIBs). In order to do so, and based on the publication by Costa et al. [7], we overexpressed GFI1, Pou4f3 and ATOH1 (hereafter referred to as GPA) coding sequences in human fibroblast cell cultures. This led to the diffe (...truncated)